Association of polymorphisms in iNOS and NQO1 with bladder cancer risk in cigarette smokers : Journal of the Chinese Medical Association

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Original Article

Association of polymorphisms in iNOS and NQO1 with bladder cancer risk in cigarette smokers

Huang, Zhon-Mina; Chen, Hsin-Anb,c; Chiang, Yi-Ted,*; Shen, Cheng-Huange,f; Tung, Min-Chea,c; Juang, Guang-Darg,h

Author Information
Journal of the Chinese Medical Association: February 2014 - Volume 77 - Issue 2 - p 83-88
doi: 10.1016/j.jcma.2013.10.005

    Abstract

    1. Introduction

    According to the annual report of the Taiwan Cancer Registry in 2012, the age-standardized incidence per 100,000 person-years of bladder cancer (BC) was 9.42 in males and 3.66 in females.1 BC is the second most common malignancy of the genitourinary tract worldwide. Cigarette smoking is the major risk factor for BC, resulting in a 2–4-fold risk among cigarette smokers.2,3 Polycyclic aromatic hydrocarbons, heterocyclic aromatic amines, and N-nitroso compounds contained in cigarettes are thought to be the carcinogenic constituents that result in the development of BC.4,5 Several chemical carcinogens contained in cigarettes were metabolized with phase II enzymes and then excreted in urine, but certain components were activated into unstable reactive oxygen species (ROS) and free radicals, leading to DNA damage.6

    NAD(P)H:quinine oxidoreductase (NQO1) is a key phase II enzyme that plays an important role in protecting cells against chemically induced oxidative stress and carcinogenicity.7 In addition, NQO1 can metabolize some chemical carcinogens such as benzo[a]pyrene-3,6-quinone, one of the carcinogenic polycyclic aromatic hydrocarbons identified in cigarettes.8 The enzyme activity may be influenced by certain functional polymorphisms located on the NQO1 gene. A common polymorphism (rs1800566) of NQO1 causes a nucleotide C-to-T transition at nucleotide 609 of exon 6 and codes for a proline (Pro)-to-serine (Ser) amino acid substitution.9,10 Previous studies have indicated that NQO1 expression is higher in bladder tumors, suggesting that NQO1 plays a key role in BC.11

    The overexpression of inducible nitric oxide synthase (iNOS) has been reported in several human cancers, including BC.12,13 iNOS can increase the nitric oxide (NO) production in response to various stimulations.14,15 It has been reported that NO production is involved in the association between inflammatory response and BC.16 Several polymorphisms in the promoter region of the iNOS gene have been shown to affect its gene expression.17,18 A highly polymorphic (CCTTT)n pentanucleotide repeat within the 5′-putative promoter region has been elucidated to modulate the expression of iNOS.19,20

    Therefore, we conducted a hospital-based case–control study to investigate the combined effect of NQO1 rs1800566 (C609T) polymorphism and iNOS (CCTTT)n pentanucleotide repeat polymorphism on the development of BC, especially among cigarette smokers in Taiwan.

    2. Methods

    2.1. Study population

    This study consisted of 309 participants enrolled from the Department of Urology, Shin-Kong Wu Ho-Su Memorial Hospital (Taipei, Taiwan) between April 2010 and June 2011. A total of 159 cases with BC (mean age, 63.9 ± 11.4 years) were diagnosed with histopathological confirmation. This was performed by board-certified pathologists using routine urological examinations. A total of 150 cancer-free controls (mean age, 62.0 ± 10.6 years) were recruited from a hospital-based pool, including those who received a general health examination at the Shin-Kong Wu Ho-Su Memorial Hospital. All participants provided informed consent prior to being interviewed and donating biospecimen. The Research Ethics Committee of the Shin-Kong Wu Ho-Su Memorial Hospital approved this study, and the study also complied with the World Medical Association Declaration of Helsinki.

    2.2. Questionnaire interview and biospecimen collection

    Demographic characteristics and lifestyle information such as cigarette smoking were collected by interviewers using a structured questionnaire. A 6–8-mL sample of vein blood was drawn from each participant for genotype determination.

    2.3. Genotyping of NQO1 rs1800566 (C609T) polymorphism

    Genomic DNA was extracted from the buffy coat of peripheral blood using the proteinase K digestion and phenol/chloroform method and then stored at −80 °C. Genotyping was determined using a polymerase chain reaction-restriction fragment length polymorphism method. Briefly, the following sequences of the primer sets were designed for the NQO1 C609T polymorphism: 5′-AAGCCCAGAC CAACTTCT-3′ (sense) and 5′-TCTCCTCATCCTGTACCTCT-3′ (antisense). The polymerase chain reaction conditions were as follows: one cycle at 95 °C for 5 minutes; 35 cycles of 95 °C for 30 seconds, 58 °C for 30 seconds, and 72 °C for 45 seconds, and a final extension at 72 °C for 7 minutes. After the digestion with the restriction enzyme HinfI (New England Biolabs), the DNA fragments that represented the NQO1 C609T polymorphism were determined (C/C: 271 bp; C/T: 271, 151, and 120 bp and T/T: 151 and 120 bp; Fig. 1). To ensure quality, a random 10% of samples were genotyped twice.

    F1-6
    Fig. 1:
    Polymerase chain reaction products were digested with restriction enzyme HinfI and analyzed by 2% agarose gel. Lane 1, 100-bp DNA ladder (MBI Fermentas); Lanes 2 and 3, C/C genotype (271 bp); Lanes 4 and 5, C/T genotype (271 bp, 151 bp, and 120 bp), and Lanes 6 and 7, T/T genotype (151 bp and 120 bp).

    2.4. Determination of iNOS (CCTTT)n pentanucleotide repeat polymorphism

    The following sequence of the primer sets were designed for the iNOS (CCTTT)n pentanucleotide repeat polymorphism: 5′-ACCCCTGGAAGCCTACAACTGCAT-3′ (sense); 5′-GCC CTGCACCCTAGCCTGTCTCA-3′ (antisense). The sense primer was labeled with FAM dye. The thermal cycling was performed with an initial step at 94° C for 5 minutes, 30 cycles of denaturation at 94° C for 1 minute, annealing at 50° C for 1 minute, and extension at 72° C for 1 minute, and a final extension at 72° C for 5 minutes. Genotyping was performed using an internal size standard by ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA).

    2.5. Statistical analysis

    A goodness-of-fit Chi-square test was used to examine the Hardy–Weinberg equilibrium (HWE) among controls. According to previous studies and the definition of the United States Centers for Disease Control, study participants who consumed more than 100 cigarettes during their lifetime were defined as ever smokers, whereas those who consumed less than 100 cigarettes in their lifetime were defined as never smokers.21–23 The combined effects of NQO1 rs1800566 (C609T) polymorphism and iNOS (CCTTT)n pentanucleotide repeat polymorphism on BC risk were estimated by odds ratios (ORs) and its 95% confidence intervals (CIs) using a multivariate-adjusted logistic regression model. All analyses were performed using SAS for Windows, version 9.1 (SAS Institute, Cary, NC, USA). A p value <0.05 was considered statistically significant.

    3. Results

    No significant differences were observed in the distribution of age, gender, and ethnicity between BC cases and cancer-free controls (Table 1). The frequency of ever smokers among BC cases (54.7%) was significant higher than that in controls (33.3%). The genotype frequency of the NQO1 C609T polymorphism in controls was in HWE (χ2 = 0.98, p = 0.52). In Table 2, study participants who carried NQO1 C/T and T/T genotypes had a significantly increased BC risk of 1.8 (95% CI = 1.1–2.9) compared to those with the C/C genotype. In addition, individuals who carried the T allele also had a significant increased BC risk of 1.4 (95% CI = 1.1–1.9) compared to those with the C allele.

    T1-6
    Table 1:
    Distribution of selected characteristics of 159 bladder cancer cases and 150 controls.
    T2-6
    Table 2:
    Distribution of NQO1 C609T polymorphism among BC cases and controls.

    The distribution of iNOS (CCTTT)n pentanucleotide repeat polymorphism is shown in Table 3. We observed four repeats (10, 11, 12, and 13) with a higher frequency (>10%) among BC cases and controls. Based on the 25th percentile of the distribution of iNOS (CCTTT)n pentanucleotide repeat polymorphism among controls, a repeat number ≤10 was regarded as short repeats (S), whereas a repeat number ≥11 was regarded as long repeats (L) in Table 4. Then, study participants were classified into three genotypes: S/S, S/L, or L/L. Compared to participants with the iNOS S/S genotype, those who carried the S/L and L/L genotypes had a nonsignificantly increased BC risk. Among ever smokers, those who carried the 12-repeat allele of the iNOS gene had a significantly increased BC risk (OR = 2.7, 95% CI = 1.0–6.7).

    T3-6
    Table 3:
    The distribution of iNOS (CCTTT)n pentanucleotide repeat polymorphism.
    T4-6
    Table 4:
    The effect of iNOS (CCTTT)n repeat polymorphism on BC risk stratified by cigarette smoking status.

    The combined effects of NQO1 C609T and iNOS (CCTTT)n polymorphisms on BC risk are shown in Table 5. After adjustment for age, gender, and cigarette smoking, participants who carried both the NQO1 C/T and T/T genotypes and the 12-repeat allele of iNOS gene had a significantly increased BC risk of 2.1 (95% CI = 1.0–4.2) compared with those who carried both the NQO1 C/C genotype and non-12-repeat allele of iNOS gene. After stratification by cigarette smoking status, the highest significant combined effect of the NQO1 C/T and T/T genotypes and the 12-repeat allele of the iNOS gene on BC was observed among ever smokers (OR = 4.4, 95% CI = 1.3–14.9).

    T5-6
    Table 5:
    The combined effects of NQO1 C609T and iNOS (CCTTT)n repeat polymorphisms on BC risk stratified by cigarette smoking status.

    4. Discussion

    BC is a multifactorial malignancy in the urinary tract system. Cigarette smoking and hazardous occupational exposure have been reported to be major risk factors for BC.24,25 Chronic exposure to chemical carcinogens contained in cigarettes will form DNA adducts and contribute to the development of various cancers, including BC.6 In the present study, we observed that BC cases had a significantly higher frequency of cigarette smoking than controls, which was consistent with previous studies.26,27

    Carcinogens contained in cigarettes usually require detoxification by phase II enzymes, leading to less toxic and more readily excreted derivatives. Therefore, the deficiency in detoxification-related enzymes cannot only cause DNA damage but also increase the accumulation of mutations.28 Genetic polymorphisms of detoxification-related enzymes have been shown to modulate the susceptibility to various malignancies.29 The NQO1 enzyme is a phase II enzyme and plays a role in the detoxification of toxic substances generated from cigarette smoking.30 In our study, significantly increased BC risk was found for participants who carried the C/T and T/T genotypes of the NQO1 gene, which was consistent with previous studies.31,32 Because the variant T allele of NQO1 C 609T polymorphism may decrease the enzyme activity in catalyzing the chemical carcinogens,7 participants who carried the C/T and T/T genotypes of the NQO1 gene were thought to be more susceptible to the development of cigarette smoking-related malignancies.

    Because NO production has been reported to be involved in the development of BC,16,33 we evaluated the effect of the iNOS (CCTTT)n repeat polymorphism on BC in a Taiwanese population. The distribution pattern of (CCTTT)n repeat numbers in the present study was similar to that reported in Japanese but not in other populations.19 Also, we found that participants carrying the S/L and L/L genotypes of the iNOS gene had a nonsignificantly increased BC risk; however, those who carried the 12-repeat allele of the iNOS gene had a significantly increased BC risk among ever smokers. The (CCTTT)n repeat polymorphism, being located upstream of the transcription start site, may affect the susceptibility to various cancers through regulating the iNOS expression.17,18 In addition, the 12-repeat allele has been found to be associated with various diseases such as colorectal cancer.34 Moreover, the distribution frequency of the 12-repeat allele was different between the Chinese population and the Caucasian population.35 Although the function of the 12-repeat allele has not been elucidated, we speculate that the iNOS 12-repeat allele could have its own effect on BC or may have to interact with functional polymorphisms located in other candidate genes. According to the combination analysis in the present study, a highest significant combined effect of the NQO1 C/T and T/T genotypes and the 12-repeat allele of the iNOS gene on BC was found among ever smokers. Therefore, our findings implied that the gene–gene interaction between the NQO1 C609T polymorphism and iNOS (CCTTT)n repeat polymorphism was predominant among cigarette smokers.

    Our study has several limitations. First, we had no detailed information about the participants' cigarette smoking habits, such as the number of cigarettes smoked per day and the number of years smoked, for investigating the association between the cumulative dosage of cigarette smoking and NQO1 C609T polymorphism and iNOS (CCTTT) repeat polymorphism. Second, clinical information regarding tumor stage and grade were not collected in this study; therefore, we could not examine the association between the tumor stage/grade and NQO1 C609T polymorphism and iNOS (CCTTT) repeat polymorphism. Third, we investigated only two polymorphisms located at NOQ1 and iNOS genes, respectively, which incompletely represent the entire function of these two genes. More functional polymorphisms should be included to validate our results in a study with a larger sample size. Therefore, the findings of our analyses must be interpreted with caution.

    In conclusion, our findings suggest there is a significant combined effect of NQO1 C/T and T/T genotypes and the 12-repeat allele of iNOS (CCTTT)n polymorphism on the development of BC, especially among cigarette smokers in Taiwan.

    Acknowledgments

    This study was supported by grants from Shin-Kong Wu Ho-Su Memorial Hospital (No. SKH-8302-99-DR-20) and Chia-Yi Christian Hospital (No. R102-7).

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    Keywords:

    bladder cancer; cigarette smoking; iNOS; NQO1; polymorphism

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